Background Various studies have discussed the benefits of applying three-dimensional (3D) techniques, specifically its advantages with respect to ergonomics, feasibility, and the rate of learning achievable in microsurgery training. However, no study has been conducted that compares the operator experience of using two-dimensional (2D) and 3D systems in microsurgical training. The aim of this study is to compare 2D- and 3D-assisted microsurgical training in novices based on anastomosis of chicken femoral arteries. Methods The participants were grouped by previous microsurgical experience. Group A includes novice participants. Group B includes 2D-experienced participants. Group C includes both participants in groups A and B. A questionnaire composed of 10 parameters in the field of image quality, dexterity, ergonomic, and feasibility will be filled out after each participant finished their anastomoses by the 2D and 3D systems. Results The results demonstrated 3D system was scored better on field of view (p = 0.004), less tremor (p = 0.005), neck/upper back comfort (p = 0.043), lower back comfort (p = 0.015), technical feasibility (p = 0.020), and educational feasibility (p = 0.004) in group A (N = 12). In group B (N = 9), 3D system was scored better on field of view (p = 0.041) but worse on image resolution (p = 0.031). Conclusion With the 3D visualization system for microsurgical anastomosis of chicken femoral model, there are significant improvements in the field of view, stability, ergonomics, and educational value compared with 2D system among all participants. Accordingly, 3D-assisted microsurgery training can be a novel and potential popular training method.